1 1 Symposium Chemosensory Receptors Satellite DEVELOPMENT ...

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165 Poster Multimodal, Chemosensory Measurement,Psychophysical, Clinical Olfactory, and TrigeminalDNA STRUCTURAL CHANGES ASSOCIATED WITH DNAHYDRATION AND ODOR POLARITY ARE MECHANISTICFACTORS IN ODOR RESPONSE BY NOVEL DNA-BASEDFLUORESCENT SENSORSWilliams L.B. 1 , White J. 1 , Kauer J. 1 1 Neuroscience, Tufts University,Boston, MAOur laboratory has developed an artificial nose that exploits 22principles of biological olfaction. Importantly, broadly tuned sensorarrays are used to achieve odor detection. In previous presentations atAChemS, we have shown that 20-30 base, solid-state, single-strandedDNA-Cy3 (ssDNA) conjugates can respond to odors. DNA-Cy3conjugates have the combinatorial potential to provide large arrays ofdifferent sensors, but their mechanism of odor response is unknown.Our experiments demonstrate that odor response in DNA-Cy3 sensorscorrelates with DNA conformational changes in a sequence and odorspecific manner. Other hypotheses (Stokes shift, pH, etc) have shownnull results. The data are consistent with DNA structural changes as theprimary modulators of Cy3 responses in DNA-Cy3 odor detection.DNA conformation appears to be altered by most odors through amechanism governed by odor polarity, although the positively chargedamines seem to have other specific interactions with DNA. In general,non-polar odors lead to ssDNA shortening and polar odors lead to anincrease in ssDNA length as measured by resonance energy transfer(FRET). FRET data also show that drying leads to changes in DNAlength similar to the effect of non-polar odors. We hypothesize that thisoccurs through odor effects on the hydration layer associated with solidstateDNA-Cy3 sensors. Changes in DNA conformation upon odorexposure may then lead to changes in Cy3 stacking with adjacent DNAbases or changes in Cy3 interactions with downstream bases, thusaltering the local environment of Cy3 and leading to a change in Cy3fluorescence. Supported by grants from NIDCD, ONR, and NSF.166 Poster Multimodal, Chemosensory Measurement,Psychophysical, Clinical Olfactory, and TrigeminalSINGLE PROTEIN NANOBIOSENSOR GRID ARRAY, IST-2001-38899-SPOT-NOSED EUROPEAN PROJECTPajot E. 1 1 European SPOT-NOSED Consortium, Institut National de laRecherche Agronomique, Jouy-en-Josas Cedex, FranceEuropean Consortium : INRA Jouy-en-Josas and Ecole Centrale deLyon, France - U. Barcelona and Campus UAB Bellaterra, Spain - U.Lecce and Politecnico di Milano, Italy The objective of SPOT-NOSEDis to develop a nanobioelectronic sensor based on the electricalproperties of single olfactory receptors anchored betweennanoelectrodes. ORs within yeast-membrane nanosomes are specificallyimmobilized onto mixed-SAM functionalized nanoelectrodes, using ananti-OR antibody and avidin-biotin interactions. Nanotransducers withnanoelectrodes were fabricated using electron beam lithography. An ORis modelled by an equivalent impedance network, predicting adetectable impedance change upon odorant binding-inducedconformational change. A transimpedance preamplifier suited for lownoisewide-bandwidth measurements of small electrical signals wasdesigned. The new instrumentation for multi-modal DC/ACmeasurements is validated in an AFM. These nanobioelectronic sensorsshould benefit from ORs individual properties : high specificity andreproducibility, low detection thresholds, large odor spectrum.Integration of individual nanosensors into multisensors arrays couldfurther increase sensitivity and widen detection spectrum, providing anew concept for powerful electronic noses/tongues mimicking in vivoodorant detection/discrimination.Numerous applications are anticipated:rapid detection/caracterization of toxic/dangerous compounds andpathological agents, food safety, medical diagnosis, follow-up ofprocesses, de-orphanization of receptors.167 Poster Multimodal, Chemosensory Measurement,Psychophysical, Clinical Olfactory, and TrigeminalUSE OF INK-JET MICRODISPENSING TO CREATECONCENTRATION-CRITICAL CHEMICAL LADEN VAPORSFOR SENSOR CALIBRATIONHayes D.J. 1 , Taylor D. 1 1 MicroFab Technologies, Inc., Plano, TXInk-jet micro-dispensing technology has been used in applicationswhere discrete picoliter volumes of fluids have been required. Theinherent precision of ink-jet based dispensing made it an ideal candidateas a calibration tool for sensor technologies. In this application, aprototype calibrator was built for the National Institute for Standardsand Technology to test if MicroFab´s microdispensing prototype candeliver explosives laden fluids precisely enough to perform as acalibration tool. The same system can be used to train dogs and testolfaction thresholds. MicroFab´s calibrator is made of threesubsystems: the PC controller, the drive electronics, and the vaporgenerator. The PC controller is simply a personal computer that runs thedrive electronics and controls the overall operation of the calibrator.MicroFab´s proprietary drive electronics contain circuitry that sends aspecific waveform to each microdispenser that subsequently ejects thecorrect drop(s). For this system, the vapor generator is made up of sixink-jet microdispensers and their reservoirs, a drop heater, and othertemperature controlling hardware. In operation, the devices inside thevapor generator delivers and vaporizes the fluid droplet. This vaporenters the ambient airflow within the vapor generator and flows to thevapor generator outlet. Maintaining the interior surfaces at an elevatedtemperature prevents vapor condensation inside the vapor generator.Testing at NIST has shown that vapor concentration of explosives canbe varied almost continuously from 0 to hundredths of parts per trillion.This range covers current standards in detection limits and will enablereal time sensor calibration.168 Poster Multimodal, Chemosensory Measurement,Psychophysical, Clinical Olfactory, and TrigeminalDEVELOPMENT OF THE OLFACT-RLHastings L. 1 , Bailie J.M. 2 1 Osmic Enterprises, Inc, Cincinnati, OH;2 Psychology, University of Cincinnati, Cincinnati, OHAssessment of olfactory detection threshold is a frequently usedclinical test for evaluating olfactory function. While administration ofthe test is time consuming and cumbersome and reliability is purportedto be low, the measure nevertheless provides important informationconcerning olfactory function. We report here the development of theOLFACT-RL test, the first economical, computer-administeredclinical olfactory detection threshold test. Stimuli are produced by anolfactometer originally developed for administering the OLFACTodor identification test. The protocol employed is similar to otherthreshold tests, e.g., CCCRC or Sniffin' Sticks, except this test iscomputer-administered. A dilution ratio of 1:2 Butanol (highestconcentration, 4%) is used to generate 16 different stimuli. Stimuli areproduced by blowing air through glass reservoirs containing theappropriate concentrations and the subject records their response via acomputer after sampling the stimulus. A single staircase with reversalparadigm is used. Three stimuli (2 blank, 1 Butanol) are presentedsequentially and the subject is asked to identify which stimulus isdifferent from the other two. If a concentration is correctly identifiedtwice in a row, the next lower concentration is presented. A misstriggers presentation of a higher concentration on the next trial. After 7reversals, threshold is determined as the mean of the last 4 reversals.Alternatively, if a miss occurs at step 1, or two consecutive hits occur atstep 16, the test is terminated. Testing of ~100 subjects resulted in afairly normal distribution of scores. Supported by DC6369 (LH)42
169 Multimodal, Chemosensory Measurement,Psychophysical, Clinical Olfactory, and TrigeminalTHE CREDIBILITY OF MEASURED ODOR THRESHOLDSSchmidt R. 1 , Cain W.S. 1 1 Surgery (Otolaryngology), University ofCalifornia, San Diego, La Jolla, CAAssume that investigators in two labs measure odor detection for thesame chemical. Each lab uses accepted methodology that differsbetween the two. One measures a threshold of 100 ppb and the other athreshold of 10 ppb. Which, if either, would you accept? All thingsbeing equal, logic says to accept 10 ppb on grounds that weaknesses inmethodology, even if unrecognized, would generally increase themeasured threshold. What if a coherent data set for more than 200materials, studied with accepted methodology and analytically verified,implied that thresholds in most compilations (US EPA, AIHA, Devos etal.) overestimated the true values, i.e., underestimated sensitivity, by anorder of magnitude? Would you accept the new results? Nagata (2002)measured such thresholds in connection with rule making in Japan.These data could make thresholds used for rule making by the US EPAand most states obsolete, as systematically too high. In experiments onsuch diverse VOCs as ethyl butyrate, hexanoic acid, limonene, andglutaraldehyde delivered via a system designed to yield stable detection,we obtained thresholds consistently below those in most compilations.Our analytically verified values lie closer to those of Nagata. Theresults imply that lower measured thresholds should generally takeprecedence over higher. They also suggest a need to rebuild the datasets that currently guide decisions regarding odor detection ofhazardous chemicals. Supported by NIH grant R01 DC05602 NIDCD.170 Poster Multimodal, Chemosensory Measurement,Psychophysical, Clinical Olfactory, and TrigeminalSELECTION OF SUBJECTS FOR CHEMOSENSORY STUDIES:CRITERIAJothi S. 1 , Cain W.S. 1 , Jalowayski A.A. 1 1 Surgery (Otolaryngology),University of California, San Diego, La Jolla, CAIt has become common to collect chemosensory data in studies ofenvironmental chemicals. It is then relevant to ask whetherdemographic variables influence outcome. Results from Woskie (1998)of chemosensory effects in workers exemplify such: “Ironically, thosewho may appear most “susceptible” to borate exposures, because oftheir greater reactivity, were the healthy non-smoking workers not usingnasal spray/drops, not reporting allergies or colds on the test day or anyhistory of bronchitis. This finding seems to contradict the commonview of susceptibility as representing compromised health status.”Whether we seek normals or others, we inevitably need to screen forhealth. We describe outcomes for screening for normal health, chronicrhinosinusitis, and allergic rhinitis. In screening for normals, a phoneinterview regarding medical history disqualified 1/6th of 299 applicants.Screening in the lab disqualified another 1/6th via interviews to confirmmedical history and to learn of current symptoms, as well as to performrhinomanometry, spirometry, examination of the eyes, nose, andoropharynx, and to collect of samples for cytological analysis. Theprincipal reason for disqualification was evidence, by one means oranother, of inflammation. In screening for the inflammatory conditionschronic rhinosinusitis and allergic rhinitis, we have added measurementof NO from the lungs and nose. From the comparison of normals withpatients with diagnosed inflammatory disease, we have come to see thatchronic inflammation can endow subjects with chemical intolerance.Supported by NIH grant DC05602 from NIDCD.171 Poster Multimodal, Chemosensory Measurement,Psychophysical, Clinical Olfactory, and TrigeminalNACL SENSATION AND HEDONICS: RELATIONSHIPS WITHSEX, TASTE GENETICS, AND SODIUM INTAKESullivan B.S. 1 , Hayes J.E. 2 , Duffy V.B. 1 1 Allied Health, University ofConnecticut, Storrs, CT; 2 Dietetics Program, University of Connecticut,Storrs, CTNaCl imparts saltiness and blocks unpleasant tastes to enhance flavor,yet sodium intakes exceed health recommendations. In 87 subjects (45men), we tested, via regression and structural equation modeling(SEM), if markers of taste variation [bitterness 6-n-propylthiouracil(PROP), fungiform papillae (FP)] explained variability in: saltiness andliking for NaCl concentration series in solution and broth; preferencefor sampled and surveyed salty foods; and salty food intake. Femalespreferred lower NaCl levels in solution or broth, regardless of PROPstatus. Men who tasted PROP as least bitter preferred the highest levels.Saltiness positively predicted liking for cheese and snack foods, butnegatively for soy sauce. Compared to supertasters, PROP nontastersliked these foods more; nontasters also tasted low sodium cheese as lessbitter and liked it more. More frequent intake of high-salt foods wasseen in those who liked high levels of NaCl in broth. SEM showedliking of sampled broth and surveyed salty food liking both predictedintake frequency of 38 salty foods, and PROP bitterness predictedintake via saltiness and liking of broth at sodium levels commonlyfound in commercial soups. Via χ2, PROP nontasters were more likelyto report `usually adding salt to food´ than were supertasters. PROPeffects on NaCl sensation may be mediated through FP number as theintensity of concentrated NaCl was associated with greater FP, notT2R38 genotype. In summary, liking for predominantly salty foodsinfluences sodium intake, with men and nontasters showing greaterliking for these foods. In foods where NaCl is added to enhance flavor,nontasters may require less to mask unpleasant tastes (NRICGP/USDA2003-35200-12943).172 Poster Multimodal, Chemosensory Measurement,Psychophysical, Clinical Olfactory, and TrigeminalDETECTION OF WEAK GUSTATORY-OLFACTORY FLAVORMIXTURESElgart B.Z. 1 , Marks L.E. 2 1 John B. Pierce Laboratory, New Haven, CT;2 Yale University, New Haven, CTPrevious research has shown that the detectability of flavor mixturescontaining olfactory and gustatory components can exceed thedetectability of either individual unmixed component. A variety ofmodels, however, can predict increased detectability, including modelsof cross-modal interaction (enhancement), cross-modal summation(summation across independent channels), and probability summationof independent channels. Using a two-alternative forced-choice methodand a sip procedure, subjects first evaluated near threshold solutions ofsucrose alone and vanillin alone at several concentrations, to establishbaseline psychometric functions for each, after which the same subjectsalso detected sucrose-vanillin mixtures. The results showed clearevidence of increased detectability of mixtures, but also suggested thepossibility of interindividual differences that may make it difficult toestablish a single general model of mixture detection. Supported byNIH Grant DC006688-0243
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